*3.5. Comparative Transcriptome Analysis of K133WT vs. K133AS-R Parasites*

Gene expression analysis using one-color DNA microarray experiment, of K133WT vs. K133AS-R isolate, revealed a modulated expression of 208 genes (approximately 2.26%) in drug-resistant parasites. The plot log<sup>2</sup> transformed expression ratio of K133AS-R (red line) vs. K133WT (green line) as a function of the chromosomal location of microarray probes is shown in Supplementary Materials Figure S1. Out of 208 differentially modulated genes, 102 genes (1.11%) were upregulated and 106 genes (1.15%) were downregulated in K133AS-R parasites. The overall expression pattern of mRNA is shown in Supplementary Materials Table S2. − .

The gene expression level on the genomic scale was analyzed using a chromosome map (Figure 5A). The chromosome map showed that chromosome 18, 25, 31, and 33 contained higher numbers of upregulated genes while chromosome 33 and 36 contained higher numbers of downregulated genes in the K133AS-R isolates. Among the upregulated genes, the highest number were present on chromosome 31, which included AQP1 (LinJ.31.0030), amastin (LinJ.31.0460, LmjF.31.0450), and a few uncharacterized proteins. Upregulated proteins include autophagocytosis protein (LinJ.33.0320), protein having RNA ligase (LinJ.33.0580) activity and transaminase (LinJ.33.1410) activity on chromosome 33, protein involved in trpanothione biosynthesis process (LinJ.18.1660) on chromosome 18, Kinesin (LinJ.25.2150), and DNA-directed RNA polymerase II (LinJ.25.1350) on chromosome 25. The maximum number of downregulated genes were present on chromosome 33, among which more than 50% were hypothetical uncharacterized proteins. Other downregulated genes on chromosome 33 included translation initiation factor 2 (LinJ.33.2880), small nuclear ribonucleoprotein complex (LinJ.33.3340), H1 histone-like protein (LinJ.33.339/0), and metallocarboxipeptidase (LinJ.33.2670). Genes showing downregulated expression on chromosome 36 included isoleucyl-t-RNA synthetase (LinJ.36.5870), translation elongation factor 1-β (LinJ.36.1490), glucose transporters (LinJ.36.6550, LmjF.36.6290, LinJ.36.6560), phosphoglycerate mutase family member 5 (LinJ.36.4270), and ubiquitin protein ligase (LinJ.36.6600).

Genes showing differential expression (both up- and downregulated genes) were classified into various functional categories and a number of altered pathways in K133AS-R parasites were identified with the help of several databases and bioinformatics tools as mentioned above in Section 2.8. The percentage of genes exhibiting altered expression with genes remained unaltered in K133AS-R parasites is shown in Figure 5B. Among the 208 genes showing modulated expression in K133AS-R parasites, a total of 144 genes were categorized into function and distributed into eight different functional categories (Figure 5C). All the 144 genes with their functional categories are enlisted in Supplementary Materials Table S3. β

**Figure 5.** *Cont*.

**Figure 5.** Comparative transcriptome profiling of K133WT and K133AS-R isolate. (**A**) Comparative gene expression of K133WT vs. K133AS-R parasites on the chromosome map. Chromosome map for differential gene expression was generated using Custom R program. Red lines indicate upregulated genes whereas green lines indicate downregulated genes in the K133AS-R parasite. (**B**) Percentage of differentially expressed genes in K133AS-R parasites. The percentage of modulated genes was calculated from the total 9170 genes obtained in Quality Control (QC) after filtering. Overall, 1.11% of genes were upregulated (red) whereas 1.15% of genes were downregulated (green); however, 97.74% of genes remained unaltered in K133AS-R parasites. (**C**) Categorization of genes showing differential expression in K133AS-R parasites according to GO functional categories. GO categories of differentially expressed genes in K133AS-R parasites suggested that genes belonging to various functional categories, such as metabolic processes, oxidation-reduction, cell membrane proteins, stress proteins, transporter activity, cell movement, and cell signaling, showed modulated expression. Unclassified proteins included hypothetical proteins with unknown function (that have not been characterized experimentally).

#### *3.6. Validation of Modulated Gene Expression Using qPCR*

Fourteen differentially expressed genes were selected for validation of expression analysis based on their role in various metabolic pathways and artesunate resistance. The selected 14 genes (9 upregulated and 5 downregulated) were validated for their expression in K133WT and K133AS-R parasites by qPCR. The fold change in the gene expression of K133AS-R/K133WT observed in q-PCR was compared with that observed in microarray experiments (Figure 6). The results obtained by qPCR for selected genes agreed with the transcriptome data derived by microarray experiments.

β **Figure 6.** Validation of modulated expression of selected genes by qPCR. Selected 14 genes showing modulated expression in a microarray were validated for their altered expression by q-PCR in three independent RNA preparations. Fold changes in the gene expression of K133AS-R parasites with respect to K133WT parasites ± SD, obtained by q-PCR and microarray experiments, are represented here. The q-PCR data were normalized using two endogenous controls, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cystathionine β-synthase (CBS).

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